Genomic and functional characterization of bacteriocinogenic lactic acid bacteria isolated from Boza, a traditional cereal-based beverage

Boza is a traditional low-alcohol fermented beverage from the Balkan Peninsula, frequently explored as a functional food product. The product is rich in Lactic Acid Bacteria (LAB) and some of them can produce bacteriocins. In this study, a sample of Boza from Belogratchik, Bulgaria, was analyzed for the presence of bacteriocinogenic LAB, and after analyses by RAPD-PCR, three representative isolates were characterized by genomic analyses, using whole genome sequencing. Isolates identified as Pediococcus pentosaceus ST75BZ and Pediococcus pentosaceus ST87BZ contained operons encoding for bacteriocins pediocin PA-1 and penocin A, while isolate identified as Pediococcus acidilactici ST31BZ contained only the operon for pediocin PA-1 and a CRISPR/Cas system for protection against bacteriophage infection. The antimicrobial activity of bacteriocins produced by the three isolates was inhibited by treatment of the cell-free supernatants with proteolytic enzymes. The produced bacteriocins inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp., among other tested species. The levels of bacteriocin production varied from 3200 to 12,800 AU/ml recorded against L. monocytogenes 104, 637 and 711, measured at 24 h of incubation at 37 °C. All bacteriocins remained active after incubation at pH 2.0–10.0. The activity mode of the studied bacteriocins was bactericidal, as determined against L. monocytogenes 104, 637 and 711. In addition, bactericidal activity was demonstrated using a cell leakage β-galactosidase assay, indicating a pore formation mechanism as a mode of action. The present study highlights the importance of combining genomic analyses and traditional microbiological approaches as way of characterizing microbial interactions in fermented foods.


Results
Screening for bacteriocinogenic LAB. The average bacterial population in the Boza sample was 1.1 × 10 8 CFU/ml recorded by plating on MRS. Colony morphology on MRS agar plates, Gram-staining and catalase test indicated that the majority of the isolates were lactic acid bacteria (LAB), with coccoid and rod morphology. Preliminary screening tests for microbial inhibition indicated that most colonies were active against Listeria monocytogenes 104. The majority presented coccid morphology and were Gram positive, catalase negative and oxidase positive. The cell-free supernatants (CFS) of 18 isolates presented antimicrobial activity against L. monocytogenes 104 and E. faecium ATCC 10434. The activity was lost after treatment of the CFS with Proteinase K and pronase, but not when treated with α-amylase, lipase or catalase, indicating the proteinaceous nature of the antimicrobial activity. Bio-molecular fingerprinting based on RAPD-PCR grouped these isolates in 3 distinct groups ( Supplementary Fig. 1). One isolate from each group was submitted to 16S rRNA sequencing for identification, resulting in Pediococcus acidilactici (one isolate) and P. pentosaceus (two isolates). These isolates were submitted to whole genome sequencing for a better characterization of the bacteriocin production.
Pediococcus acidilactici ST31BZ genome characterization. The genome assembly for strain ST31BZ produced 19 contigs with N50 of 433,517 bp and 235 × coverage. MiGA essential genes analysis showed a completeness of 94.6%, contamination of 1.8%, with an overall quality of 85.6%. We detected 105 of 111 essential genes in this genome and it was classified by MiGA using genome-aggregate Average Nucleotide and Amino Acid Identity (ANI/AAI) concepts as P. acidilactici, and closely related with P. acidilactici ZPA017 (NZ_CP015206.1) (98.89% ANI), isolated from black pig in Beijing, China.
Pediococcus acidilactici ST31BZ genome presented genes coding for several sugar transporters such as sucrose, maltose/glucose and cellobiose, and is likely able to degrade them all to lactate and acetate. We also found evidence that the strain is able to synthesize arginine, glycine, alanine, serine, asparagine, aspartate, glutamine and glutamate (Supplementary Table 1).
Pediococcus pentosaceus ST75BZ and ST87BZ genome characterization. The assembled genomes of the two P. pentosaceus isolates presented a very high similarity (99.99% ANI), despite being analyzed separately, the only difference being that we obtained 15 contigs with N50 of 282,292 bp and 63 × genome coverage for isolate ST75BZ, and 15 contigs with N50 of 296,046 bp and 62 × genome coverage for isolate ST87BZ. The following results apply to both isolates and we will refer to them as strain P. pentosaceus ST75BZ and ST87BZ for the remainder of the text (Fig. 1b).
MiGA essential genes analysis showed a completeness of 95.5%, contamination of 1.8% and an excellent quality of 86.5% for both isolates. We detected 106 of 111 essential genes and this strain was taxonomically classified by MiGA, using genome-aggregate ANI/AAI concepts, as P. pentosaceus, and closely related with P. pentosaceus ATCC 25745 (NC_008525.1) (99.1% ANI). The genome size for P. pentosaceus ST75BZ and ST87BZ isolate was 1,810,333 bp, average GC content was 37.08%, with 1773 protein-coding genes predicted and no pseudogenes. www.nature.com/scientificreports/ containing a pediocin PA-1 operon (Fig. 2b). Other pediocin-like bacteriocin operon found was annotated as penocin A (Fig. 2c), present in the strain's genome. We also found point mutations in the 23S rRNA coding sequence predicted to confer resistance to macrolides (azithromycin) and streptogramins using ARIBA. Using KEGG GhostKoala pipeline, other resistance genes were also detected: Beta lactamase class A gene (PenP), a Cationic Antimicrobial Peptide (CAMP) resistance gene, the dltABCD operon (Dlt A/B/C/D), two Multidrug efflux pump genes (EfrA/B and AbcA); and a lincosamide resistance gene (Lsa). The genome of P. pentosaceus ST75BZ and ST87BZ presented similar values for size, GC content, number of predicted proteins and ribosomal operons as 65 strains of P. pentosaceus in China 11 . Strains P. pentosaceus ST75BZ and ST87BZ genome presented similar metabolic capabilities as P. acidilacti ST31BZ, however this strain has a reduced sugar transport capability, is able to synthesize lysine and is positive for genes encoding for a quorum sensing mechanism that we were not able to attribute to a distinct physiologic state or metabolic process (Supplementary Table 2). Pangenomic analysis of Pediococcus spp.. Pangenomic analysis of the Pediococcus spp. isolates was carried out using Anvi' o v6.1 12 with the pangenomic workflow. We selected five complete genomes of P. acidilactici and compared to P. acidilactici ST31BZ genome (Fig. 3a). Pediococcus acidilactici ST31BZ strain formed a related group with P. acidilactici ZPA017 (98.89% ANI) and BCC1 (98.84% ANI). We found 1487 core genes (1360 Single-copy Core genes, SCG) in all six genomes. Pediococcus acidilactici ST31BZ has 152 unique genes (Supplementary Table 3). COG annotation indicated prediction of six genes that encode phage-related proteins, in addition to six genes related to transposase activity, and 15 genes involved in carbohydrate metabolism, including a Na +/melibiose symporter and an arabinose efflux permease.
Based on the pangenomes of our isolates against reference genomes, we propose P. pentosaceus ST31BZ as a new strain of P. acidilactici and strains ST75BZ and ST87BZ as a new strain of P. pentosaceus. The two www.nature.com/scientificreports/ closest strains to P. acidilactici were ZPA017 and BCC-1, isolated from feces of a healthy pig 13 and cecum of a broiler chicken 14 , respectively. The unique genes present in our isolates were related to antibiotic resistance and prophages, such as glycopeptide antibiotics resistance protein (COG4767) and phage portal protein BeeE (COG4695) in P. acidilactici ST31BZ (Supplementary Table 3); and ABC-type multidrug transport system (COG0842) and phage terminase large subunit (COG1783) in P. pentosaceus ST75BZ and ST87BZ. Pediococcus pentosaceus ST75BZ and ST87BZ, also showed the presence genes related to SOS response system, such as SOSresponse transcriptional repressor (COG1974) (Supplementary Table 4). Our two novel isolates presented pediocin PA-1 coding genes in their plasmids, which were highly similar (96% identity). Additionally, P. pentosaceus ST75BZ and ST87BZ presented a penocin A gene in its genome. Our annotation pipeline also detected bacteriocin genes in some of the strains used in the pangenome analysis: we detected two enterolyzin A genes in the genome of P. acidilactici BCC1, a bovicin coding gene in P. acidilactici BCC1 plasmid; an enterolysin A gene in the genome of P. acidilactici ZPA017; and a penocin A gene and enterolysin A gene in the genome of P. pentosaceus ATCC 25745.
Bacteriocin production kinetics and stability tests. For P. acidilactici ST31BZ, P. pentosaceus ST75BZ and P. pentosaceus ST87BZ, only a small amount of bacteriocins were detected in the cell surfaces (approximately 200 AU/ml), with the majority of activity detected in cell-free culture supernatants. All remaining assays were carried out using cell-free supernatants, unless otherwise stated.
We characterized the bacteriocins produced by our three selected isolates for their stability in different pH, salt concentration, temperature and detergents. The three isolates grew well when cultured in MRS at 25, 30 and 37 °C for 24 h, and produced bacteriocins (12,800 AU/ml for ST31BZ and 3200 AU/ml for ST75BZ and ST87BZ). All further experiments were performed at 37 °C, taking in consideration potential future applications of these strains as probiotics for human application. The activity of the bacteriocins remained unaltered after exposure to different pH (from 4.0 to 10.0), temperature (10,25,30,37,45,80, 100 °C for up to 240 min, and at 121 °C for 15 min), or in the presence of NaCl, skim milk, SDS, Tween 20 and Tween 80 and EDTA, highlighting their potential versatility for use in industrial settings (data not shown).
The end-pH recorded for P. acidilactici ST31BZ, P. pentosaceus ST75BZ and P. pentosaceus ST87BZ when cultured overnight in MRS at 37 °C were 4.43, 4.1 and 4.05, respectively (Fig. 4a). Over the same time period (27 h), the cell density increased from approximately OD 600nm 0.04 to 3.03 for P. acidilactici ST31BZ, 0.08 to 4.7 for P. pentosaceus ST75BZ and 0.06 to 4.77 for P. pentosaceus ST87BZ (Fig. 4a). Moreover, levels of bacteriocin produced by P. acidilactici ST31BZ were gradually increased during the fermentation process and reached 12,800 AU/ml at 15 h and remained stable until the end of the monitored period of 27 h (Fig. 4a). However, bacteriocin produced by P. pentosaceus ST75BZ reached its maximum production levels (6400 AU/ml) at 15 h from the beginning of fermentation, remained stable until 18 h of fermentation time and decreased in the next 9 h (Fig. 4b). As expected, a similar bacteriocin production profile was recorded for both P. pentosaceus strains (Fig. 4c).    4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Time ( teriocins produced by P. acidilactici ST31BZ, P. pentosaceus ST75BZ or P. pentosaceus ST87BZ was investigated using a growth inhibition assay, by adding 6400 AU/ml of bacteriocins produced by the strains to early-logarithmic growing (3-h-old) cultures of L. monocytogenes 104, 637 and 711. All three novel strains inhibited the growth of Listeria (Fig. 5), and recovery of viable L. monocytogenes from the cultures after 10 and 24 h of growth was not possible, indicating the killing effect. We investigated cell membrane pore formation using a β-galactosidase leakage assay. Cells of L. monocytogenes 104, 637 and 711 were incubated for 10 min with cell-free supernatants containing 6400 AU/ml of bacteriocin, followed by the addition of the β-galactosidase substrate and colorimetric product read-out. All bacteriocin/Listeria combinations tested produced a positive result, indicating that the mechanism of action involves pore formation and the destabilization of the cell membrane on the test strains. Antibiotic susceptibility. From 20 different tested antibiotics, all P. acidilactici ST31BZ, P. pentosaceus ST75BZ, and P. pentosaceus ST87BZ were resistant to 10 of them (ampicillun, amikacin, ciprofloxacin, gentamicin, kanamycin, metronidazole, oxacillin, tetraciclin, tobramycin, and nalidix acid). In addition, P. acidilactici ST31BZ and P. pentosaceus ST75BZ showing resistance to vancomycin (complete list in Supplementary  Table 6).

Discussion
Boza is a traditional fermented beverage rich in nutrients, presenting a diverse microbial composition. Tests with Boza samples from different regions of the Balkan peninsula have shown that LAB and yeasts are the main microorganisms involved in the fermentation 1,2 . The present study aimed to characterize the potential the bacteriocin production by strains isolated from a sample of Boza obtained from a medium scale manufacture in North-west Bulgaria, using a mixed molecular and functional approach.
The screening method allowed us to select 3 bacterial isolates, initially classified as Pediococcus sp. Genome analysis indicated that these three isolates likely belong to two different Pediococcus species: one P. acidilactii, and two P. pentosaceus. The P. acidilactici ST31BZ genome showed similar values of size, GC content, number of predicted proteins and ribosomal operons as other P. acidilactici strains 13,15 . Detected CRISPR systems are similar to previously described in P. acidilactici HN9 10 . A small plasmid-encoded bacteriocin (~ 9 kb) was detected, such as found in pSMB74 16 and pCP289 17 . Several studies have shown that strains of P. pentosaceus are able to produce bacteriocins 6 , and in our study we observed the presence of two pediocin-like bacteriocins, pediocin PA-1 and penocin A, both of which have been previously reported in P. pentosaceus genomic analyses. Antibiotic resistance, prophages, plasmid and plasmid-associated bacteriocins have been indicated as source of variability at P. pentosaceus genomes 11 . Our P. acidilactici strain, however, also contains a plasmid-associated bacteriocin operon (pediocin PA-1), differing from other strains reported in the literature, such as ZPA017 and BCC-1 13,14 . The metabolic characteristics of the strains indicated a wider metabolic potential for carbohydrate transport for  www.nature.com/scientificreports/ the P. acidilactii transport while the P. pentosaceus presented he capability to synthesize lysine and had quorum sensing related genes. Data from our initial screening indicated that the antimicrobial metabolites produced by the studied strains were proteinaceous in nature, which prompted us to further characterize the potential mechanisms of action and the activity range for the bacteriocins being produced. Although a peptide or protein molecule must be present for the antimicrobial activity to be detected, this observation does not preclude the possibility that other moieties may also be present in a larger complex that has the final bacteriocinogenic effect, as previously reported [18][19][20] . LAB can produce a variety of antimicrobial compounds, including diacetyl, hydrogen peroxide, carbon dioxide, low molecular antimicrobial substances, and organic acids such as phenyl lactic acid 18 . It is possible that other Enterococcus faecalis (20) Enterococcus faecium (13)

Listeria innocua (5)
Listeria monocytogenes (38) Pediococcus spp. (6) S. infantarius subsp. infantarius (2) Salmonella spp. (8) Staphylococcus aureus (14) Staphylococcus epidermidis (4) Streptococcus thermophilus (11)  www.nature.com/scientificreports/ protein or peptide-derived antibiotics could be produced by these strains, as we have detected a gene signature for lantibiotic resistance in the genome of the P. acidilactici strain. However, based on the obtained results, we can clearly exclude the possibility of acid/s or H 2 O 2 to be involved in the antimicrobial properties of the studied strains.
Identification of the genes involved in bacteriocin biosynthesis is an important part of the characterization of the antimicrobial agent as bacteriocin(s). Bacteriocin genes can be part of the bacterial genome, and their expression and detection in the cell free supernatant need to be confirmed. Therefore, a functional approach is important in evaluating the bacteriocin's activity. Bacteriocins produced by the three selected strains presented a wide range of activity and were thermostable, maintaining activity after exposure to different temperatures up to 4 h, including at 121 °C for 20 min. Such activity breadth is highly desirable for industrial applications. Most bacteriocins are molecules smaller than 10 kDa, although some can form complexes of higher molecular weight 21 . This small size confers stability in harsh conditions. Other reports have described that bacteriocins produced by different Pediococccus spp. are stable at different pH and after exposure to low and high temperatures 22 . Surfactants and salts normally have not been reported to negatively influence bacteriocins activity 23 , however, plantaricin C19 activity was affected by treatment with SDS or Triton X-100 24 . SDS did not cause loss of activity as has been observed for enterocin EJ97, bozacin B14 or bacteriocin ST194BZ 22,25 . However, pH can play a role on the stability of the bacteriocins, as been reported for leucocin F10 26 . Moreover, lactocin NK24 stability was decreased when exposed to 100 °C for 30 min and even was completely inactivated at 121 °C for 15 min 27 . Similar effect of temperature was reported for lactocin MMFII, produced by L. lactis MMFII 28 . Even nisin, one of the best studied bacteriocins, was shown to be inactivated after 15 min at 121 °C when incubated at pH 7.0, but not when incubated at pH 3.0 29 .
We observed a strong antimicrobial activity of P. acidilactici ST31BZ, P. pentosaceus ST75BZ and P. pentosaceus ST87BZ against a wide range of bacteria tested. These strains produce bacteriocins with strong antimicrobial activity recorded against all Listeria strains and almost all enterococci evaluated. Nevertheless, no antimicrobial activity was observed against S. aureus, Lactobacillus spp. (except Latilactobacillus sakei), Leuconostoc spp., Samonella spp. and other Pediococcus spp. strains tested. Specific anti-Listeria activity is an attribute reported for different studied pediocins and particularly pediocin PA-1. Even Heng et al. 21 in his classification of bacteriocins, dedicated a special position for anti-Listeria pediocin-like bacteriocins. This specific activity was linked to the conservative amino acid motive directly involved in the bacteriocin mode of action and pore formation process 21 .
There are reports in the literature that some bacteriocins can be adsorbed onto the producer's cell surface, and this characteristic can be used to facilitate the purification process and/or increase the bacteriocin yield for industrial application 30 . In case of the studied bacteriocins, only low levels of cell-adsorption were recorded, which cannot be considered relevant in increasing production yield. Similar results have been shown for pediocins produced by P. acidilactici HA-6111-2 and HA-5692-3, respectively, against L. innocua N27 and E. faecium HKLHS 23 . Moreover, pediocin PA-1 has been reported as an effective bactericidal bacteriocin for the control of different L. monocytogenes strains, leading to is proposition as prospective biopreservation for different fermented food products 23,31,32 . Furthermore, the results obtained from the β-galactosidase leakage assay confirm that bacteriocins produced by P. acidilactici ST31BZ, P. pentosaceus ST75BZ and P. pentosaceus ST87BZ likely destabilize the cell membrane inducing the cytoplasmic leakage and a complete loss of viability for the susceptible strains (data not shown). Similar approach and results have been reported for bacteriocins produced by Lactiplantibacillus plantarum, Le. lactis and E. faecium 18 , Lentilactobacillus buchneri 33 , Lactiplantibacillus plantarum 34,35 and Lc. lactis subsp. lactis 36 .
This study confirms previous ones that have shown boza to be a rich source of LAB of biotechnological and industrial interest. The bacteriocinogenic potential of the isolated P. acidilactici ST31BZ, P. pentosaceus ST75BZ and P. pentosaceus ST87BZ strains reported here include a partial characterization of the mechanism of action and breadth of susceptible bacterial targets. As most of the fermented traditional food products, Boza is a rich multimicrobial system, were interactions between different microbial species is essential for the final product's characteristics. Results of whole genome sequencing analysis of the three selected bacteriocinogenic strains, coupled with functional assays, highlight their metabolic potential for industrial application, including production at industrial level of Boza or any other fermented functional food product.

Materials and methods
Screening for bacteriocinogenic lactic acid bacteria in boza. Boza sample was obtained from a medium-scale manufacturer in North-west Bulgaria. Isolation of bacteriocinogenic lactic acid bacteria (LAB) from these samples was done according to the 3-layer method 37 . Samples of boza were submitted to serial decimal dilutions in sterile saline (0.85% NaCl, Sigma Diagnostics, St. Louis, MO, USA) and plated on the surface of MRS agar (Difco BD, Franklin Lakes, NJ, USA) plates. After addition of a layer of 1.0% agar (Difco), the plates were incubated for 48 h at 37 °C. The colonies were counted and plates with well isolated colonies were added of an extra layer of BHI agar (Difco) (BHI supplemented with 1.0% agar) containing L. monocytogenes 104 or E. faecium ATCC 19434 (10 5 CFU/ml). Plates were incubated for additional 24 h and colonies with visible inhibition zones were transferred to new BHI and incubated at 37 °C for 24 h. The cultures were checked for purity by streaking on MRS agar. Individual colonies on MRS agar were submitted to Gram-staining and catalase and oxidase tests. Presumed bacteriocin producing LAB and other microorganisms used as target organisms were stored at − 80 °C in MRS or BHI added of 20% glycerol.

Identification of the antimicrobial metabolite produced by the selected strains. The isolates
were grown in MRS broth at 37 °C for 24 h, and submitted to centrifugation (6000 × g, 10 min, 4 °C) for obtention of cell free supernatants (CFS). After adjustment of the pH to 5.0-6.5 with 1 M NaOH, the CFS were heated www.nature.com/scientificreports/ for 10 min at 80 °C to eliminate potential inhibitory effect of organic acids and to inactivate hydrogen peroxide and proteolytic enzymes in the medium. Ten microliters of treated CFS were spotted on the surface on plates containing BHI supplemented with 1.0% agar plates containing 10 5 CFU/ml L. monocytogenes 104 or E. faecium ATCC 19434, used as target test micro-organisms. Plates were incubated at 37 °C for 24 h and presence of growth inhibition zones wider than 2 mm was considered evidence for potential bacteriocin production (Supplementary Table 5). The proteinaceous nature of the antimicrobial substances produced by the isolates was investigated in the cell free supernatants by treatment with Proteinase K, pepsin and pronase (all from Sigma), as described before 37 . Antimicrobial activity and the effect of temperature (30,60,120  The obtained reads were processed with the software package BBTools (https:// jgi. doe. gov/ data-and-tools/ bbtoo ls/). Reads were trimmed for Nextera adapters and filtered to have an average Q-score < 15. Trimmed reads from the three strains were assembled de novo with SPAdes 3.13.0 40 . ORFs were predicted for all assembled contigs with Prokka pipeline 41 . Scaffolds were uploaded to MiGA to access genome assembly completeness and to identify the closest bacterial strain to each assembled genome 42 . tRNA were detected with ARAGORN 43 and rRNAs with Barrnap (https:// github. com/ tseem ann/ barrn ap). CRISPR regions were detected with PILER-CR 44 and CRISPRCasFinder 45 . Plasmid searches were made using metagenomic plasmid function (metaplasmidspades. py) 46 and the presence of plasmid genes verified using the script viralVerify 47 with -p argument. Bacteriocins genes were detected with BAGEL4 48 and antibiotic resistances genes using ARIBA 49 with quality filtered reads and CARD database 50 .
Pangenomic analysis of Pediococcus isolates were done using anvi' o v6.1 12 with the pangenomic workflow. Our objective was to compare the similarity of isolates and reference genomes. We focused on P. acidilactici and P. pentosaceus species. All sequencing data generated in this study can be accessed from GenBank Database at BioProject PRJNA731169. The genome of isolate ST31BZ was compared to five reference genomes of P. acidilactici species available on NCBI (BioProject numbers: PRJNA312971, PRJNA357663, PRJNA422477, PRJNA386762, and PRJNA386761). The genomes of isolates ST75BZ and ST87BZ were compared to four references genomes of P. pentosaceus species available on NCBI (PRJNA399825, PRJNA398, PRJNA376813, and PRJNA390207).

Spectrum of activity.
The selected bacteriocinogenic strains were grown in MRS at 37 °C for 24 h and cell free supernatants were obtained as described before. Inhibitory effectiveness of the produced bacteriocins was evaluated against several LAB, selected food borne pathogens and some Gram-negative organisms, listed in Supplementary Table 5. The growth conditions (culture medium and temperature) were according to the recommended for each test microorganism. Test microorganisms were grown overnight and incorporated in appropriate medium, supplemented with 1.0% agar, at final concentration around 10 5 CFU/ml. Studied bacteriocins were spotted (10 μl) on the surface and plates cultured for 24 h at recommended growth temperature. Zones of inhibition, larger than 2 mm were considered as positive result.
Growth and bacteriocin production dynamics. The selected strains were grown in 20 ml MRS broth (Difco) at 25 °C, 30 °C or 37 °C for 24 h. Cell free supernatant was prepared as described before and bacteriocin activity determined against L. monocytogenes 104 and expressed in AU/ml. After selection the optimal temperature for bacteriocin production, the dynamic of the production was evaluated as follows: overnight cultures were prepared in 300 ml MRS broth (Difco) at 37 °C, and optical density at 600 nm and pH changes were monitored hourly for 24 h. Production of bacteriocin(s) was measuring the antimicrobial activity against L. monocytogenes 104, 637 and 711 every three hours (expressed as AU/ml). Experiments were performed in two independent occasions. www.nature.com/scientificreports/ Inhibitory effect of bacteriocins evaluated via cell lysis assay. For evaluation of the effect of studied bacteriocin on actively growing L. monocytogenes 104, 637 and 711, the approach proposed by de Castilho et al. 51 was followed. For the experiment, 300 ml BHI broth was inoculated with 1% (v/v) of L. monocytogenes 104, 637 or 711 and incubated for 3 h at 37 °C. 30 ml filter-sterilized (0.22 μm Millipore sterile filters, Burlington, MA, USA) cell-free supernatant of each bacteriocinogenic strain was added to the culture and changes on the turbidity were monitored at OD 600nm every hour for 10 h. In addition, bacterial growth was determined after 24 h of incubation, to check for viable cells. The numbers of CFU/ml were determined after 10 h and 24 h of cultivation for all assays, i.e., with added bacteriocin and controls (without added bacteriocin), by plating on BHI supplemented with 2% agar and incubation at 37 °C for 48 h. Experiments were performed in duplicate in 2 independent occasions.
β-Galactosidase assay. The capability of bacteriocin(s) to induce pore formation in the target strains was tested determining the level of β-galactosidase secreted from damaged cells 36 . Cells from 20 ml of actively growing, log-phase cultures of L. monocytogenes 104, 637 and 711 were harvested, washed twice with 20 ml 0.03 M sodium phosphate buffer (K 2 HPO 4 /KH 2 PO 4 , pH 6.5) and the pellet re-suspended into 10 ml of the same buffer. Two ml of the cell suspensions were treated with equal volumes of each studied bacteriocin to yield final concentrations of 6 400 AU/ml. After 10 min at 37 °C, 0.

Data availability
Genomes assembled and their SRA sequencing data are available in the NCBI under BioProject PRJNA731169.